Revealing the Mechanisms and Physiology of Peroxisome Transfer in Stem Cell Therapy

揭示干细胞治疗中过氧化物酶体转移的机制和生理学

• 批准号: 9889194
• 负责人: Troy C. Lund
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9889194
• 关键词: Adoptive Transfer; Adrenal Glands; Adrenoleukodystrophy; Animal Model; Brain; Cell Communication; Cell Line; Cell Separation; Cell physiology; Cells; Cellular biology; Cerebrum; Cessation of life; Child; Childhood; Coculture Techniques; Communication; Connexins; Data; Defect; Disease; Dose; Flow Cytometry; Generations; Genes; Goals; Hematopoietic; Immunohistochemistry; In Vitro; Ink; Knowledge; Label; Lead; Life; Lipid Binding; Liver; Marrow; Measures; Metabolism; Microscopy; Mitochondria; Modeling; Monitor; Morbidity - disease rate; Mus; Mutation; Nanotubes; Organ; Organelles; Peroxisomal Disorders; Pharmaceutical Preparations; Physiological; Physiology; Process; Proteins; Reactive Oxygen Species; Stromal Cells; Structure; System; Time; Tissues; Transgenic Mice; Transplantation; Very Long Chain Fatty Acid; Vesicle; Wild Type Mouse; Zebrafish; autocrine; boys; cell type; central nervous system demyelinating disorder; cytokine; design; early childhood; exosome; experimental study; hematopoietic cell transplantation; high reward; high risk; in vivo; male; novel; novel therapeutics; oxidation; paracrine; peroxisome; protein function; small molecule; stem cell therapy; success; tool; uptake

Project Summary (Abstract). Cell-to-cell communication is important in all tissues and organs and occurs by a variety of mechanisms. Cytokine secretion, gap junction proteins, small molecules, and exosomes have been shown to facilitate the exchange of cytoplasmic “information” between cells. Another novel mode of cellular communication is that of organelle transfer which has been shown to occur with mitochondria. Peroxisomes are small lipid bound organelles which are responsible for the metabolism of very long chain fatty acids in the cell. There are several rare, but devastating childhood diseases that are a result of peroxisomal defects such adrenoleukodystrophy (ALD). We have new evidence that peroxisomes can be transferred between cell types. We hypothesize that peroxisomal transfer could be a mechanism that allows for the stabilization of cells harboring dysfunctional peroxisomes. The goal of this application is to develop an animal model in which peroxisomes are labeled within hematopoietic cells to allow adoptive transfer experiments and observation of peroxisomal transport in vitro and in vivo. Aim 1: Optimize the zebrafish model of peroxisomal transfer. Our pilot data, generated in a zebrafish model of hematopoietic transplant, indicate that donor cells can transfer peroxisomes to recipient hematopoietic cells. It would be ideal to understand if we can fully optimize this system in terms of cell dose and time after HCT. We will determine the optimal conditions for peroxisome transfer to occur in this model by varying the cell doses and time after transplant readout. Transfer will be determined by flow cytometry and verified by cell sorting and fluorescent microscopy. Aim 2: Study the in vitro phenomenon of peroxisomal transfer between various cell types and monitor changes in cellular function. We have created a variety of cell lines and have primary cells with GFP containing a peroxisome tag sequence (PTS-GFP) which allows for specific peroxisome labeling. We will perform co-culture experiments to observe peroxisomal transfer and will quantify the physiological effects of transferring healthy peroxisomes into cells with dysfunctional peroxisomes measuring reactive oxygen species and mitochondrial function. Aim 3: Generation of a PTS-GFP expressing mouse under the control of the Rosa locus. The goal of this aim is the establishment of a colony of PTS-GFP transgenic mice to be used as donors or recipients in adoptive experiments allowing us to determine if peroxisomal transfer occurs in vivo. HSPC from donor mice will transplanted into wild-type mice. PTS-GFP mice can also be used as recipients to determine if cells of the marrow niche can transfer peroxisomes into unlabeled donor cells. This project carries both high risk and high reward, as its success will open an entirely new field and thought process into peroxisomes, organelle transfer, and cellular communication. We will gain new knowledge and build tools to not only better understand peroxisomal transfer, but develop new strategies, drugs, and small molecules to enhance the process which could impact how we treated devastating peroxisomal disease like ALD.

项目摘要（摘要）。细胞间的通讯在所有组织和器官中都很重要